Crystalline genistein sodium salt dihydrate

ABSTRACT

The disclosure relates to a new crystalline form of genistein. The disclosed crystalline form is crystalline genistein sodium salt dihydrate. The disclosure also relates to the novel genistein salt composition represented by this crystalline form. Therapeutic compositions containing crystalline genistein sodium salt and a pharmaceutically acceptable carrier are described. The disclosure also relates to therapeutic methods comprising the step of administering to a patient in need thereof a therapeutically effective amount of a therapeutic composition containing the crystalline form of the disclosure or crystalline genistein sodium salt dihydrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application61/121,778, filed Dec. 11, 2008, which is incorporated herein byreference.

BACKGROUND

Cancer is characterized by uncontrolled cell growth which occurs whenthe normal regulation of cell proliferation is lost. This loss oftenappears to be the result of dysregulation of the cellular pathwaysinvolved in cell growth and division, apoptosis, angiogenesis, tumorinvasion and metastasis.

Genistein,4′,5,7-trihydroxyisoflavone-5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one,(shown below), is a natural compound present in plants such as soy.Genistein's potential role in the prevention and treatment of a numberof human diseases

including cancer has been extensively studied. Genistein is a BCS classII isoflavone that is commercially available from a number of sourcesincluding LC Laboratories, Woburn, Mass. The cellular targets forgenistein and the signaling pathways regulated by genistein have beenidentified and those related to cancer include targets and pathwaysimportant for cell growth and division, apoptosis, angiogenesis, tumorinvasion and metastasis. In addition to the inherent anti-tumor effectsof genistein itself, studies have shown that genistein also potentiates,or accentuates, the anti-tumor effects of several clinically usedchemotherapeutic agents both in vitro in human cancer cell lines and invivo in animal models of cancer. From a therapeutic perspective, thesedata are interesting as chemotherapy is the cornerstone in the treatmentof most solid tumors.

Genistein is practically insoluble in water but has high cell membranepermeability. Low water solubility and slow dissolution rate are oftenlimiting factors responsible for the low bioavailability ofpharmaceutical compounds, limiting their application.

Despite the long known fact that genistein has certain properties ofanti-cancer drugs, no successful genistein treatment regimens have been,or are, employed in the treatment of cancers. One plausible explanationfor this is probably the poor solubility and poor bioavailability aswell as the rapid phase II metabolism of genistein in its known form.

Due to the development of the drug discovery strategy over the last 20years, physicochemical properties of drug development candidates havechanged significantly. The development candidates are generally morelipophilic and less water soluble, which creates huge problems for theindustry. Research has shown that some drug candidates fail in theclinical phase due to poor human bioavailability and problems with theformulation. Traditional methods to address these problems, withoutcompletely redesigning the molecule, include salt selection, producingamorphous material, particle size reduction, pro-drugs, and differentformulation approaches. Recently, crystalline forms of activepharmaceutical ingredient (API) have been used to alter thephysicochemical properties of the API.

Although therapeutic efficacy is the primary concern for a therapeuticagent, the salt and solid state form (i.e., the crystalline or amorphousform) of a drug candidate can be critical to its pharmacologicalproperties and to its development as a viable API. For example, eachsalt or each crystalline form of a drug candidate can have differentsolid state (physical and chemical) properties. The differences inphysical properties exhibited by a novel solid form of an API (such as acocrystal, salt, or polymorph of the original compound) affectpharmaceutical parameters such as storage stability, compressibility anddensity (important in formulation and product manufacturing), andsolubility and dissolution rates (important factors in determiningbioavailability). Because these practical physical properties areinfluenced by the solid state properties of the crystalline form of theAPI, they can significantly impact the selection of a compound as anAPI, the ultimate pharmaceutical dosage form, the optimization ofmanufacturing processes, and absorption in the body. Moreover, findingthe most adequate polymorphic form for further drug development canreduce the time and the cost of that development.

Obtaining crystalline forms of an API is extremely useful in drugdevelopment. It permits better characterization of the drug candidate'schemical and physical properties. It is also possible to achieve desiredproperties of a particular API by forming a salt of the API and/or acrystalline salt of the API. Crystalline forms and crystalline saltsoften have better chemical and physical properties than the free base inits amorphous state. Such salts and crystalline forms may, as with thepresent invention, possess more favorable pharmaceutical andpharmacological properties or be easier to process than the amorphouspolymorphic form. They may also have better storage stability.

One such physical property, which can affect processability, is theflowability of the solid, before and after milling. Flowability affectsthe ease with which the material is handled during processing into apharmaceutical composition. When particles of the powdered compound donot flow past each other easily, a formulation specialist must take thatfact into account in developing a tablet or capsule formulation, whichmay necessitate the use of glidants such as colloidal silicon dioxide,talc, starch or tribasic calcium phosphate.

Another potentially important solid state property of an API is itsdissolution rate in aqueous fluid. The rate of dissolution of an activeingredient in a patient's stomach fluid may have therapeuticconsequences since it impacts the rate at which an orally administeredactive ingredient may reach the patient's bloodstream.

By forming and/or crystallizing a salt of an API, a new solid state formof the API may have unique properties compared with existing solid formsof the API or its salt. For example, a crystalline salt may havedifferent dissolution and solubility properties than the API itself andcan be used to deliver APIs therapeutically. New drug formulationscomprising crystalline salts of APIs may have superior properties overexisting drug formulations.

A crystalline salt or other crystalline form of an API generallypossesses distinct crystallographic and spectroscopic properties whencompared to other forms having the same chemical composition.Crystallographic and spectroscopic properties of the particular form aretypically measured by X-ray powder diffraction (XRPD) and single crystalX-ray crystallography, among other techniques. Particular crystallineforms often also exhibit distinct thermal behavior. Thermal behavior ismeasured in the laboratory by such techniques as capillary meltingpoint, thermogravimetric analysis (TGA) and differential scanningcalorimetry (DSC).

SUMMARY

The invention relates to crystalline genistein sodium salt dihydrate.Therapeutic compositions containing the crystalline genistein sodiumsalt dihydrate of the invention represents another embodiment of theinvention, as do methods of treating cancer and other hyperproliferativediseases with that crystalline salt or therapeutic compositionscontaining it. Therapeutic compositions of crystalline genistein sodiumsalt dihydrate may also be used for the treatment of chronicinflammation, infection, cystic fibrosis and amyloidosis.

As used herein and as known in the art, the term “ambient temperature”means a temperature within an enclosed space at which humans areaccustomed, i.e., room temperature. For example, ambient temperature mayrange, for example, from about 20° C. to about 25° C.

As used herein and as known in the art, the term “approximately” meansnear to in quantity or amount.

As used herein and as known in the art, the term “slurry” means asuspension of solids in a liquid.

As used herein and as known in the art, the term “°2θ” isinterchangeable with [degree-two-theta], [°2Th.], and variationsthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures, which are described below and which areincorporated in and constitute a part of the specification, illustrateexemplary embodiments according to the disclosure and are not to beconsidered limiting of the scope of the invention, for the invention mayadmit to other equally effective embodiments. The figures are notnecessarily to scale, and certain features and certain views of thefigures may be shown exaggerated in scale or in schematic in theinterest of clarity and conciseness.

FIG. 1 depicts an XRPD pattern of crystalline genistein sodium saltdihydrate.

FIG. 2 depicts a DSC trace of dried crystalline genistein sodium saltdihydrate.

FIG. 3 depicts a gravimetric vapor sorption (GVS) trace of crystallinegenistein sodium salt dihydrate.

FIG. 4 depicts a TGA trace from a sample of prepared crystallinegenistein sodium salt dihydrate that was dried at ambient temperaturefor about 24 hours.

FIG. 5 depicts a TGA trace from a sample of prepared crystallinegenistein sodium salt dihydrate that was dried at 80° C. overnight.

FIG. 6 depicts four XRPD patterns of crystalline genistein sodium saltdihydrate taken after stability studies at 80° C. for 7 days and at 40°C./75 relative humidity (RH) % for 7 days.

FIG. 7 is a ¹H nuclear magnetic resonance (NMR) spectra of crystallinegenistein sodium salt dihydrate.

FIG. 8 depicts XRPD patterns after a hydration study of crystallinegenistein sodium salt dihydrate.

FIG. 9 is a molecular model of crystalline genistein sodium saltdihydrate, illustrating the centrosymmetric disodium cation in thedimeric structure of crystalline genistein sodium salt dihydrate,wherein the intramolecular hydrogen bonds are shown as dashed lines.

FIG. 10 is a molecular model illustrating a layer formation ofcrystalline genistein sodium salt dihydrate.

FIG. 11 is a molecular model illustrating a packing of crystallinegenistein sodium salt dihydrate.

FIG. 12 depicts a calculated XRPD pattern based on single crystal datafor crystalline genistein sodium salt dihydrate.

FIG. 13 depicts the plasma concentration of total genistein afterintraduodenal administration of genistein and crystalline genisteinsodium salt dihydrate (mean, n=3).

FIG. 14 depicts an XRPD pattern for crystalline genistein sodium saltdihydrate from the large scale synthesis.

FIG. 15 depicts an XRPD pattern for crystalline genistein.

DETAILED DESCRIPTION

The current invention relates to improvements of the physiochemicalproperties of genistein, whereby this compound may be suitable for drugdevelopment. Disclosed herein is a new crystalline form of genistein, acrystalline genistein sodium salt dihydrate Although a crystalline saltof genistein is described herein, the invention also relates to thenovel chemical composition genistein sodium salt dihydrate. Thetherapeutic uses of the crystalline form is described as well astherapeutic compositions containing it The methods used to characterizethe crystalline forms are also described below.

One embodiment of the invention relates to a crystalline genisteinsodium salt dihydrate. The crystalline genistein sodium salt dihydratemay possess suitable characteristics for pharmaceutical development. Theonly possible negative may be its needle-like morphology which is notnecessarily ideal for flowability or compression during manufacture. Theneedle-like morphology was observed using Polarized Light Microscopy(PLM). Milling of this crystalline needle-like material, or similartechniques known in the art, may be used to achieve more uniformparticle morphology, which may be used to prepare the material formanufacturing its pharmaceutical composition. One of ordinary skill candetermine particle sizes appropriate for a desired pharmaceuticalcomposition. Particle sizes of about 5 μm, for example, may be used. Itshould be noted, however, that sustained milling may dehydrate thematerial due to the high temperatures involved during such processes. Onthe other hand, the 80° C. storage tests have indicated that thematerial can exist as a hydrate at elevated temperatures over a 7 dayperiod with only a slight change. This mitigates the risk of dehydrationon milling.

As shown in FIG. 9, the crystalline genistein sodium salt dihydrate ofthe invention has a dimeric structure centrosymmetric disodium cation inassociation with two genistein molecules and four water molecules. Thecrystalline genistein sodium salt dihydrate may be prepared from, forexample, IPA (propan-2-ol or isopropanol), a common solvent, at ambienttemperature without the need for any special treatment such astemperature cycling, sonication or rapid evaporation. As shown inExample 1 below, the crystalline genistein sodium salt dihydrate of theinvention possesses excellent stability. It is more soluble in water,aqueous solvent systems and organic solvents than genistein itself. Inaddition, the crystalline genistein sodium salt dihydrate shows superiorearly and late intrinsic kinetic solubility profiles as compared togenistein. The crystalline genistein sodium dihydrate of the inventionhas also been shown to have greater bioavailability than genistein.

Therapeutic Uses of the Crystalline Forms of Genistein

The invention relates to therapeutic uses of crystalline genisteinsodium salt dihydrate. The term “treatment” or “treating” means anytreatment of a disease or disorder in a mammal, including: preventing orprotecting against the disease or disorder, that is, causing theclinical symptoms not to develop; inhibiting the disease or disorder,that is, arresting or suppressing the development of clinical symptoms;and/or relieving the disease or disorder, that is, causing theregression of clinical symptoms. It will be understood by those skilledin the art that in human medicine, it is not always possible todistinguish between “preventing” and “suppressing” since the ultimateinductive event or events may be unknown, latent, or the patient is notascertained until well after the occurrence of the event or events.Therefore, as used herein the term “prophylaxis” is intended as anelement of “treatment” to encompass both “preventing” and “suppressing”as defined herein. The term “protection,” as used herein, is meant toinclude “prophylaxis.”

The crystalline form of genistein according to the invention may beuseful as a medicament, which may be used to treat hyperproliferativediseases such as, for example, various cancers, including, for example,colorectal, gastric, esophageal, breast, lung, prostate, bladder, brain,renal, ovarian, liver, skin, thyroid, and pancreatic cancer, as well asleukemias or lymphomas. The leukemias and lymphomas mentioned herein maybe tumors of myeloid lineage such as, for example, acute myeloidleukemia or of lymphoid lineage.

Additionally, the crystalline form of genistein disclosed herein mayalso be used in a method of treatment of a warm-blooded animal such as,for example, man, by therapy. For example, the crystalline genisteinsodium salt dihydrate according to the invention may be useful in amethod of treatment of hyperproliferative diseases such as, for example,various cancers, including, for example, colorectal, gastric,esophageal, breast, lung, prostate, bladder, brain, renal, ovarian,liver, skin, thyroid, and pancreatic cancer, as well as leukemias orlymphomas. The leukemias and lymphomas mentioned herein may be tumors ofmyeloid lineage such as, for example, acute myeloid leukemia or oflymphoid lineage.

Moreover, the crystalline form of genistein according to the inventionmay be used in the method of treating a human suffering from ahyperproliferative disease such as, for example, various cancers,including, for example, colorectal, gastric, esophageal, breast, lung,prostate, bladder, brain, renal, ovarian, liver, skin, thyroid, andpancreatic cancer, as well as leukemias or lymphomas. In anotherembodiment, the crystalline form of genistein according to the inventionmay be used to prevent a hyperproliferative disease such as, forexample, various cancers, including, for example, colorectal, gastric,esophageal, breast, lung, prostate, bladder, brain, renal, ovarian,liver, skin, thyroid, and pancreatic cancer, as well as leukemias orlymphomas. The leukemias and lymphomas mentioned herein may be tumors ofmyeloid lineage, such as, for example, acute myeloid leukemia or oflymphoid lineage, comprising the steps of administering to a person inneed thereof a therapeutically effective amount of the crystalline formof genistein according to the invention. The use of the crystalline formof genistein in any of the methods of treating a human described abovealso form aspects of this invention.

The treatment defined herein may be applied as a sole therapy or mayinvolve, in addition to the compound of the invention, conventionalsurgery or radiotherapy or chemotherapy. Such chemotherapy may includeone or more of the following categories of anti-tumor agents: (i)antiproliferative/antineoplastic drugs and combinations thereof, as usedin medical oncology, such as alkylating and alkylating-like agents (forexample, cis-platin, carboplatin, cyclophosphamide, nitrogen mustard,melphalan, chlorambucil, busulphan and nitrosoureas), antimetabolites(for example, gemcitabine HCl, 5-fluorouracil, tegafur, raltitrexed,methotrexate, cytosine arabinoside and hydroxyurea), antitumorantibiotics (for example, anthracyclines like adriamycin, bleomycin,doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin and mithramycin), antimitotic agents (for example, vincaalkaloids like vincristine, vinblastine, vindesine and vinorelbine andtaxoids like taxol and taxotere), and topoisomerase inhibitors (forexample, epipodophyllotoxins like etoposide and teniposide, amsacrine,topotecan and camptothecin); (ii) cytostatic agents, such asantioestrogens (for example, tamoxifen, toremifene, raloxifene,droloxifene and iodoxyfene), oestrogen receptor antagonists (forexample, fulvestrant), antiandrogens (for example, bicalutamide,flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRHagonists (for example, goserelin, leuprorelin and buserelin),progestogens (for example, megestrol acetate), aromatase inhibitors (forexample, anastrozole, letrozole, vorazole and exemestane), andinhibitors of 5-alpha-reductase (for example, finasteride); (iii) agentswhich inhibit cancer cell invasion (for example, metalloproteinaseinhibitors like marimastat and inhibitors of urokinase plasminogenactivator receptor function); (iv) inhibitors of growth factor function,for example, such inhibitors include growth factor antibodies, growthfactor receptor antibodies (for example, the anti-ErbB2 antibodytrastuzumab (Herceptin), and the anti-ErbB1 antibody (cetuximab)),farnesyl transferase inhibitors, tyrosine kinase inhibitors, andserine-threonine kinase inhibitors, for example, inhibitors of theepidermal growth factor family (for example, EGFR family tyrosine kinaseinhibitors such asN-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine(gefitinib, AZD1 839),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib, OSI-774) and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)qumazolin-4-amine(CI 1033)), inhibitors of the platelet-derived growth factor family, andinhibitors of the hepatocyte growth factor family; (v) antiangiogenicagents such as those which inhibit the effects of vascular endothelialgrowth factor (for example, the anti-vascular endothelial cell growthfactor antibody bevacizumab (Avastin) and compounds such as thosedisclosed in International Patent Applications WO 97/22596, WO 97/30035,WO 97/32856 and WO 98/13354) and compounds that work by other mechanisms(for example, linomide, inhibitors of integrin function andangiostatin); (vi) vascular damaging agents such as Combretastatin A4and compounds disclosed in International Patent Applications WO99/02166, WO 00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and WO02/08213; (vii) antisense therapies, for example, those which aredirected to the targets listed above, such as ISIS 2503, an anti-rasantisense; (viii) gene therapy approaches, including, for example,approaches to replace aberrant genes such as aberrant p53 or aberrantBRCA1 or BRCA2, GDEPT (gene-directed enzyme pro-drug therapy) approachessuch as those using cytosine deaminase, thymidine kinase or a bacterialnitroreductase enzyme and approaches to increase patient tolerance tochemotherapy or radiotherapy such as multi-drug resistance gene therapy;and (ix) immunotherapy approaches, including for example, ex-vivo and invivo approaches to increase the immunogenicity of patient tumor cells,such as transfection with cytokines such as interleukin 2, interleukin 4or granulocyte-macrophage colony stimulating factor, approaches usingtransfected immune cells such as cytokine-transfected dendritic cells,approaches using cytokine-transfected tumor cell lines, and approachesusing anti-idiotypic antibodies.

In the treatment discussed above, the crystalline form of genisteinaccording to the invention may also be used in combination with one ormore cell cycle inhibitors, for example, with cell cycle inhibitorswhich inhibit cyclin-dependent kinases (CDK), or in combination withimatinibmesylate (Glivec). Such joint treatment may be achieved by wayof the simultaneous, sequential or separate dosing of the individualcomponents of the treatment. Such combination products may employ thecompound of this invention within the dosage range described herein andthe other at least one pharmaceutically-active agent within its approveddosage range. Combination products may be formulated into a singledosage form.

The invention also provides a combination that may be suitable for usein the treatment of cell proliferative disorders (such as cancer)comprising the crystalline form of genistein of the invention and atleast one additional anti-tumor agent as defined hereinbefore. Suchcombination may serve as a pharmaceutical product for the conjointtreatment of cell proliferative disorders (such as cancer).

In addition to their use in therapeutic medicine, at least onecrystalline form of genistein according to the invention may also beuseful as pharmacological tools in the development and standardizationof in vitro and in vivo test systems for the evaluation of the effectsof inhibitors of cell cycle activity in laboratory animals such as cats,dogs, rabbits, monkeys, rats and mice, as part of the search for newtherapeutic agents.

Another aspect of invention relates to therapeutic uses of thecrystalline form of genistein according to the invention in thepreparation of a medicament for the treatment of a disease where theinhibition of inflammation is beneficial, such as, for example, chronicinflammation, inflammatory bowel disease, Crohn's disease, Sjögren'sdisease, rheumatoid arthritis, arthritis, atopic dermatitis, vasculitis,psoriasis, benign prostate hyperplasia, wound healing, end stage renaldisease, chronic kidney disease, chronic obstructive pulmonary disease,or asthma.

Additionally, the crystalline form of genistein according to theinvention may also be used in the preparation of a medicament for thetreatment of a disease where the inhibition of infection is beneficial,such as, for example, local infection, systemic infection, sepsis,systemic fungal infection, or local fungal infection.

Yet another aspect of the invention relates to the use of thecrystalline form of genistein for the treatment of a disease whererestoring normal chloride and salt (water) movements in body organs andpeople's glands is beneficial, such as, for example, stimulating thecystic fibrosis transmembrane conductance regulator.

Yet another aspect of the invention relates to the use of thecrystalline form of genistein for the treatment of a disease whereinhibition of a soluble protein from forming insoluble extracellularfibril deposits causing organ dysfunction is beneficial, such as, forexample, inhibition of transthyretin (TTR) amyloidoses caused byalterations in the amino acid sequence of the TTR gene product. Inanother embodiment of the disclosure, the crystalline form of genisteindescribed herein may be used for the treatment of Familial AmyloidPolyneuropathy.

Pharmaceutical Compositions Containing Crystalline Forms of Genistein

The invention also relates to pharmaceutical compositions comprising atherapeutically effective amount of the crystalline form of genisteinaccording to the invention and a pharmaceutically acceptable carrier(also known as a pharmaceutically acceptable excipient). As discussedabove, the crystalline forms of genistein according to the invention maybe therapeutically useful for the treatment or prevention of, forexample, the disease states discussed above, including, for example,those associated with abnormal angiogenesis.

Pharmaceutical compositions for the treatment of those disease statesmay contain a therapeutically effective amount of the crystalline formof genistein according to the invention to down-regulate thetranscription of genes involved in controlling angiogenesis fortreatment of a patient with the particular disease. A pharmaceuticalcomposition of the invention may be in any pharmaceutical form whichcontains the crystalline form of genistein according to the invention.The pharmaceutical composition may be, for example, a tablet, capsule,liquid suspension, injectable, topical, or transdermal. Thepharmaceutical compositions generally contain, for example, about 1% toabout 99% by weight of the crystalline form of genistein of theinvention and, for example, 99% to 1% by weight of at least one suitablepharmaceutical excipient. In one embodiment, the composition may bebetween about 5% and about 75% by weight of the crystalline form ofgenistein of the invention with the rest being at least one suitablepharmaceutical excipient or at least one other adjuvant, as discussedbelow.

A “therapeutically effective amount of the crystalline genistein sodiumsalt dihydrate according to the invention” is generally in the range ofabout 0.050-about 500 mg/kg. The actual amount required for treatment ofany particular patient may depend upon a variety of factors including,for example, the disease state being treated and its severity; thespecific pharmaceutical composition employed; the age, body weight,general health, sex and diet of the patient; the mode of administration;the time of administration; the route of administration; and the rate ofexcretion of the crystalline form of genistein; the duration of thetreatment; any drugs used in combination or coincidental with thespecific compound employed; and other such factors well known in themedical arts. These factors are discussed in Goodman and Gilman's “ThePharmacological Basis of Therapeutics”, Tenth Edition, A. Gilman, J.Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001, which isincorporated herein by reference. The crystalline form of genisteinaccording to the invention and pharmaceutical compositions containing itmay be used in combination with anticancer or other agents that aregenerally administered to a patient being treated for cancer. They mayalso be co-formulated with one or more of such agents in a singlepharmaceutical composition.

Depending on the type of pharmaceutical composition, thepharmaceutically acceptable carrier may be chosen from any one or acombination of carriers known in the art. The choice of thepharmaceutically acceptable carrier depends upon the pharmaceutical formand the desired method of administration to be used. For apharmaceutical composition of the invention, that is one having thecrystalline form of genistein of the invention, a carrier should bechosen that maintains the crystalline form. In other words, the carriershould not substantially alter the crystalline form of genistein. Norshould the carrier be otherwise incompatible with the crystalline formof genistein used, such as by producing any undesirable biologicaleffect or otherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition.

The pharmaceutical compositions of the invention may be prepared bymethods know in the pharmaceutical formulation art, for example, seeRemington's Pharmaceutical Sciences, 18th Ed., (Mack Publishing Company,Easton, Pa., 1990), which is incorporated herein by reference. In asolid dosage form, the crystalline form of genistein may be admixed withat least one pharmaceutically acceptable excipient such as, for example,sodium citrate or dicalcium phosphate or (a) fillers or extenders, suchas, for example, starches, lactose, sucrose, glucose, mannitol, andsilicic acid, (b) binders, such as, for example, cellulose derivatives,starch, alignates, gelatin, polyvinylpyrrolidone, sucrose, and gumacacia, (c) humectants, such as, for example, glycerol, (d)disintegrating agents, such as, for example, agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, croscarmellosesodium, complex silicates, and sodium carbonate, (e) solution retarders,such as, for example, paraffin, (f) absorption accelerators, such as,for example, quaternary ammonium compounds, (g) wetting agents, such as,for example, cetyl alcohol, and glycerol monostearate, magnesiumstearate and the like (h) adsorbents, such as, for example, kaolin andbentonite, and (i) lubricants, such as, for example, talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, or mixtures thereof. In the case of capsules, tablets, andpills, the dosage forms may also comprise buffering agents.

Pharmaceutically acceptable adjuvants known in the pharmaceuticalformulation art may also be used in the pharmaceutical compositions ofthe invention. These include, but are not limited to, preserving,wetting, suspending, sweetening, flavoring, perfuming, emulsifying, anddispensing agents. Prevention of the action of microorganisms may beensured by inclusion of various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, and the like. Itmay also be desirable to include isotonic agents, for example, sugars,sodium chloride, and the like. If desired, a pharmaceutical compositionof the invention may also contain minor amounts of auxiliary substancessuch as wetting or emulsifying agents, pH buffering agents,antioxidants, and the like, such as, for example, citric acid, sorbitanmonolaurate, triethanolamine oleate, butylated hydroxytoluene, etc.

Solid dosage forms as described above may be prepared with coatings andshells, such as enteric coatings and others well known in the art. Theymay contain pacifying agents, and can also be of such composition thatthey release the active compound or compounds in a certain part of theintestinal tract in a delayed manner. Non-limiting examples of embeddedcompositions that may be used are polymeric substances and waxes. Theactive compounds may also be in microencapsulated form, if appropriate,with one or more of the above-mentioned excipients.

Suspensions, in addition to the active compounds, may contain suspendingagents, such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,or mixtures of these substances, and the like.

Compositions for rectal administrations are, for example, suppositoriesthat may be prepared by mixing the crystalline genistein sodium saltdihydrate according to the invention with, for example, suitablenon-irritating excipients or carriers such as cocoa butter,polyethyleneglycol or a suppository wax, which may be solid at ordinarytemperatures but may be liquid at body temperature and, therefore, meltwhile in a suitable body cavity and release the active componenttherein.

Because the crystalline form is maintained during preparation, soliddosage forms are preferred for the pharmaceutical composition of theinvention. Solid dosage forms for oral administration, which includescapsules, tablets, pills, powders, and granules, may be used. In suchsolid dosage forms, the active compound may be mixed with at least oneinert, pharmaceutically acceptable excipient (also known as apharmaceutically acceptable carrier). The crystalline genistein sodiumsalt dihydrate according to the invention may also be used as precursorsin the formulation of liquid pharmaceutical compositions. Administrationof this crystalline form of genistein in pure form or in an appropriatepharmaceutical composition may be carried out via any of the acceptedmodes of administration or agents for serving similar utilities. Thus,administration may be, for example, orally, buccally, nasally,parenterally (intravenous, intramuscular, or subcutaneous), topically,transdermally, intravaginally, intravesically, intrasystemically, orrectally, in the form of solid, semi-solid, lyophilized powder, orliquid dosage forms, such as, for example, tablets, suppositories,pills, soft elastic and hard gelatin capsules, powders, solutions,suspensions, or aerosols, or the like, such as, for example, in unitdosage forms suitable for simple administration of precise dosages. Oneroute of administration may be oral administration, using a convenientdaily dosage regimen that can be adjusted according to the degree ofseverity of the disease-state to be treated.

The invention also relates to preparation of a medicament using thecrystalline genistein sodium salt dihydrate for the treatment of avariety of diseases. These include, but are not limited to: diseaseswhere the inhibition of one or more protein tyrosine kinase(s) isbeneficial, such as, for example, kinases which are affected bygenistein are possible targets; hyperproliferative diseases such asvarious cancers, such as, for example, colorectal, breast, lung,prostate, bladder, renal or pancreatic cancer, or leukemia or lymphomaor proliferative inflammatory atrophy; diseases where the inhibition ofinflammation is beneficial, such as, for example, chronic inflammation,inflammatory bowel disease, Crohn's disease, Sjögren's disease,rheumatoid arthritis, arthritis, atopic dermatitis, vasculitis,psoriasis, benign prostate hyperplasia, wound healing, end stage renaldisease, chronic kidney disease, chronic obstructive pulmonary disease,asthma; diseases where the inhibition of infection is beneficial, suchas, for example, local infection, systemic infection, sepsis, systemicfungal infection, local fungal infection; diseases where restoringnormal chloride and salt (water) movements in body organs and glands inpeople is beneficial, such as, for example, stimulating the cysticfibrosis transmembrane conductance regulators as well as diseases andsymptoms relating to postmenopausal condition such as hot flushes andosteoporosis as well as diseases where inhibition of a soluble proteinfrom forming insoluble extracellular fibril deposits causing organdysfunction is beneficial, such as amyloidosis, for example, those wherethe fibril deposits are composed of Transthyretin (TTR), such asFamilial Amyloid Polyneuropathy.

EXAMPLES

The following analytical techniques were used in the examples below:

X-ray Powder Diffraction (XRPD): X-ray powder diffraction studies wereperformed on a Bruker D8-Discover diffractometer. Approximately 5 mg ofsample was gently compressed on the XRPD zero back ground single 96 wellplate sample holder. The sample was then loaded into a BrukerD8-Discover diffractometer in transmission mode and analyzed using theexperimental conditions shown in Table 1.

TABLE 1 XRPD Measurement Conditions Raw Data Origin BRUKER-binary V3(.RAW) Scan Axis Gonio Start Position [°2Th.] 4.0000 End Position[°2Th.] 49.9800 Step Size [°2Th.] 0.0200 Scan Step Time [s] 39.1393 ScanType Continuous Offset [°2Th.] 0.0000 Divergence Slit Type FixedDivergence Slit Size [°] 2.0000 Specimen Length [mm] 10.00 ReceivingSlit Size [mm] 0.1000 Measurement Temperature [° C.] 25.00 AnodeMaterial Cu K-Alpha1 [Å] 1.54060 K-Alpha2 [Å] 1.54443 K-Beta [Å] 1.39225K-A2/K-A1 Ratio 0.50000 Generator Settings 40 mA, 40 kV DiffractometerNumber 0 Goniometer Radius [mm] 250.00 Dist. Focus-Diverg. Slit [mm]91.00 Incident Beam Monochromator No Spinning No

Differential Scanning Calorimetry (DSC): Approximately 2 mg of samplewas weighed into an aluminum DSC pan and sealed with an aluminum lid(non-hermetically). The sample pan was then loaded into a Pyris 1Perkin-Elmer DSC (equipped with a liquid-nitrogen cooling unit) cooledand held at 25° C. Once a stable heat-flow response was obtained, thesample was then heated to 300° C. at a scan rate of 10° C./min and theresulting heat flow response monitored. A 20 cm³/min helium purge wasused to prevent thermally induced oxidation of the sample during heatingand also to reduce the thermal lag through the sample to increase theinstrument sensitivity. Prior to analysis, the instrument wastemperature and heat-flow calibrated using an indium reference standard.

Gravimetric Vapor Sorption (GVS): Approximately 15 mg of sample wasplaced into a wire-mesh vapor sorption balance pan and loaded into anSMS intrinsic vapor sorption balance supplied (Surface MeasurementSystems Instruments). The sample was then dried by maintaining a 0%humidity environment until no further weight change was recorded.Subsequently, the sample was then subjected to a ramping profile from0-90% RH at 10% RH increments, maintaining the sample at each step untilequilibration had been attained (99.5% step completion). Upon reachingequilibration, the % RH within the apparatus was ramped to the next stepand the equilibration procedure repeated. After completion of thesorption cycle, the sample was then dried using the same procedure. Theweight change during the sorption/desorption cycles were then monitored,allowing for the hygroscopic nature of the sample to be determined.

Thermogravimetric Gravimetric (TGA): Approximately 5 mg of sample wasaccurately weighed into a platinum TGA pan and loaded into aPerkin-Elmer TGA 7 gravimetric analyzer held at room temperature. Thesample was then heated at a rate of 10° C./min from 25° C. to 300° C.during which time the change in weight monitored. The purge gas used wasnitrogen at a flow rate of 20 cm³/min. Prior to analysis, the instrumentwas weight calibrated using a 100 mg reference weight and temperaturecalibrated using an alumel reference standard.

Polarized Light Microscopy (PLM): The presence of crystallinity(birefringence) was determined using a Leica Leitz DMRB polarizedoptical microscope equipped with a high resolution Leica camera andimage capture software (Firecam V.1.0). All images were recorded using10× objectives unless otherwise stated.

¹H Nuclear Magnetic Resonance (NMR): ¹H NMR was performed on a BrukerAC200. NMR of each sample was performed in deutero-methanol. Each samplewas prepared in ca. 5 mg concentration.

Example 1 Crystalline Genistein Sodium Salt Dihydrate

1.1 Preparation of Genistein Sodium Salt Dihydrate: ca., 300 mg ofgenistein was placed in 6 cm³ (20 vols) of IPA. On addition of 1M sodiumhydroxide (NaOH) a reaction was quickly evident (color change from paleyellow to vibrant yellow). The mixture was allowed to shake at ambienttemperature for ca. 3 hrs and then allowed to stand over ca. 2 days(weekend). The solid was isolated by filtration and allowed to dry atambient temperature for ca. 24 hrs. The genistein sodium salt preparedaccording to this method is crystalline genistein sodium salt dihydratewhich has been characterized by the following methods.

1.2 XRPD of Crystalline Genistein Sodium Salt Dihydrate

The XRPD pattern as shown in FIG. 1 was obtained using the proceduredescribed above. As shown in FIG. 1, the XRPD analysis reveals a solidform impurity which is probably an IPA solvate of the sodium salt.Drying the material at 80° C. overnight removes the impurity. The peaksin the XRPD pattern at an experimental °2θ±0.2 °2θ are listed in Table2. The entire list of peaks, or a subset thereof, may be sufficient tocharacterize crystalline genistein sodium salt dihydrate. One subset ofpeaks that, individually or in combination, may be used to characterizecrystalline genistein sodium salt dihydrate from FIG. 1 includes 5.9,11.6, 11.8, 15.2, 24.8, 28.1, 28.9, and 29.0 °2θ±0.2 °2θ.

TABLE 2 Pos. d- [°2θ ± 0.2 spacing Rel. Int. °2θ] [Å] [%] 5.4 16.5 6.255.9 15.0 14.05 7.5 11.8 21.61 8.2 10.8 4.19 11.6 7.6 6.4 11.8 7.5 7.4214.8 6.0 4.3 15.2 5.8 71.86 16.0 5.5 5.13 16.8 5.3 8.89 17.1 5.2 7.3123.5 3.8 5.6 24.3 3.7 22.41 24.8 3.6 100 25.5 3.5 7.31 26.7 3.3 4.8727.0 3.3 5 28.2 3.2 17.86 28.5 3.1 22.31 28.9 3.1 14.37 29.7 3.0 4.35

1.3 DSC of Dried Crystalline Genistein Sodium Salt Dihydrate

A sample was prepared by drying the crystalline genistein sodiumdihydrate salt, prepared according to the procedure described in 1.1above, at 80° C. overnight. FIG. 2 shows the DSC of the sample of driedcrystalline genistein sodium salt dihydrate. The DSC indicatesdehydration ca. 91° C. followed by melting at ca. 132° C. The otherpeaks are probably associated with degradation (as also indicated by theTGA traces shown in FIGS. 4 and 5, discussed below).

1.4 GVS of Crystalline Genistein Sodium Salt Dihydrate

As shown in FIG. 3, the GVS study of crystalline genistein sodiumdihydrate indicated hydrate formation (GVS cycle dehydrates materialprior to analysis) and a maximum of 45 wt % of water adsorbed. However,between 20 and 70 RH % (typical working range of material) only ca. 2%moisture change was observed.

1.5 TGA of Crystalline Genistein Sodium Salt Dihydrate

FIG. 4 shows the TGA trace from a sample of crystalline genistein sodiumsalt dihydrate that was dried at ambient temperature for about 24 hours,1.1 above. FIG. 5 is a TGA from a sample of prepared crystallinegenistein sodium dihydrate salt that was dried at 80° C. overnight. TheTGA indicates that the sodium salt is hydrated and water loss commencesat ca. 75° C., suitable for further development. The weight loss isconsistent with one mole of water to one mole of sodium.

1.6 PLM of Crystalline Genistein Sodium Salt Dihydrate

The PLM of crystalline genistein sodium salt dihydrate showed aneedle-like morphology.

1.7 Solubility Measurements of Crystalline Genistein Sodium SaltDihydrate

Aqueous Solubility: Aqueous solubility was measured using the followingprotocol. Slurries of genistein and of crystalline genistein sodiumdihydrate salt were made up in aqueous media in which pH was set at 4.5,6.7, and 7.5, each slurry was shaken at ambient temperature for ca. 24hours and then filtered using a 0.2 μm filter into a clean vial. Thesaturated solutions were then diluted and analyzed for API (genistein)content using N-Ac-DL-Methionine on a Chirobiotic T HPLC column and UVdetector set at λmax=270 nm. The mobile phase was acetonitrile/water runin isocratic mode over a 30 minute period. The results are presented inTable 3, (BDL=below detection limits). No API peaks were evident fromthe HPLC traces run with genistein (should appear at ca. 6-7 min)indicating that genistein is extremely insoluble in aqueous media andthat the levels are below the sensitivity of the HPLC technique employed(sensitivity of the technique mg to μg level). Genistein is reported toexhibit an aqueous solubility in the range of 10-40 nM.

TABLE 3 Crystalline Genistein Genistein, Sodium Salt Dihydrate, Solventmg/ml mg/ml Water/slurry pH 4.5 BDL 0.136 Water/slurry pH 6.7 BDL 0.707Water/slurry pH 7.5 BDL 0.650

Solubility in different solvents: Solubility in different organicsolvents was measured using the following protocol. Approximately 25 mgportions of genistein and crystalline genistein sodium dihydrate saltwere placed in 48 different vials, separately. 5 volume aliquots of eachsolvent were added exclusively to a vial. Between each addition, themixture was checked for dissolution and if no dissolution was apparent,the procedure was continued until dissolution was observed or when 50volumes had been added. The results are shown in Table 4.

TABLE 4 Crystalline Genistein Genistein Sodium Salt Dihydrate SolventSolubility, mg/ml Solubility, mg/ml Methanol (MeOH) <10.6 <8.8 Ethanol(EtOH) <10.8 98 2-Propanol (IPA) <10.5 <10.2 Acetone/IPA (50:50) 20.120.2 1-Butanol (BuOH) <10.5 19.3 Methyl acetate (MeOAc) <10.3 <10.2Acetone ca. 17 <10.2 1,4-dioxane <10.3 <10.6 Acetonitrile (MeCN) <10.1<10.4 Tetrahydrofuran (THF) 26.3 <10.4 Dichloromethane (DCM) <10.3 <10.8tert-Butylmethyl ether <10.5 <9.2 (TBME) Methylethyl ketone (MEK) <10.8<9.2 Heptane <10.5 <9.9 Octanol <10.5 <11 N-N-dimethylformamide ca.100 >217.6 (DMF) Dimethyl sulfoxide (DMSO) ca. 100 >235.2 Toluene <10.1<9.9 N-Methyl-2-pyrrolidinone ca. 68 90.4 (NMP) Methyl isobutyl ketone<10.1 <9.0 (MIBK) Acetone/Water (50:50) <10.1 69.6 Toluene/Dioxane(50:50) <10.1 <9.7 Cyclohexane <10.7 <10.9 Diisopropylether (DIPE) <10.6<9.7

1.8 Stability Study of Crystalline Genistein Sodium Salt Dihydrate

Sample stability was tested at 80° C. for 7 days and at 40° C./75 RH %for 7 days. Observations such as color change were noted after 7 daysand XRPD of samples were taken after 7 days to investigate any solidform change. FIG. 6 shows the XRPD patterns of the original sample andsamples of crystalline genistein sodium salt dihydrate at 80° C. for 7days and at 40° C./75 RH % for 7 days. The 40° C./75 RH % studyindicated no change over a 7 day period. Storing the material at 80° C.over a 7 day period indicated a slight loss in crystallinity suggestingslow dehydration. The 7 day light stability tests revealed no change incolor or solid form.

1.9 ¹H NMR Spectrum of Crystalline Genistein Sodium Salt Dihydrate

FIG. 7 illustrates the ¹H NMR spectrum of the crystalline genisteinsodium salt dihydrate. Table 5 lists the peaks in the ¹H NMR spectrum.Displacement of the chemical shifts for the aromatic protons at ca. 5.9in genistein to 6.1 ppm in the ¹H NMR of FIG. 8 confirms salt formation.

TABLE 5 Chemical Shift Multiplicity Range (ppm) 7.952 s 7.932-7.9197.372 m 7.429-7.306 6.861 m 6.927-6.791 6.101 dd 6.187-6.028 4.936 s5.256-4.723 3.34 q 3.577-3.096 1.085 s 1.213-0.934 s = singlet, m =multiplet, dd = doublet of doublet, q = quadruplet

1.10 Disproportionation Study of Crystalline Genistein Sodium SaltDihydrate

A 50 mg sample of crystalline genistein sodium salt dihydrate wasslurried in 250 μL distilled water for ca. 48 hours and then checked byXRPD for disproportionation. The pH of the supernatant was also measuredusing a Corning 240 pH meter. No signs of disproportionation wereobserved. The pH of the supernatant after slurrying was 7.1.

1.11 Hydration Study of Crystalline Genistein Sodium Salt Dihydrate

Approximately 100 mg of crystalline genistein sodium salt dihydrate wereplaced in ca. 500 μL IPA/water mixtures (3%, 5% and 10%) at the waterlevel. Each mixture was agitated for ca. 48 hours at ambient temperatureand then filtered to recover the solid for XRPD and TGA studies. Asshown in FIG. 8, hydration was indicated from a change from the originalmaterial in the XRPD pattern corresponding with weight loss from TGA(material depending). The hydration study revealed no further hydrates;but removed the IPA solvate impurity.

1.12 Single Crystal X-ray Diffraction of Crystalline Genistein SodiumSalt Dihydrate

Single Crystal Preparation: Crystals were grown from solutions ofcrystalline genistein sodium salt dihydrate (ca. 48 mg) dissolved in50:50 IPA/Water (3 cm³). The solution was then allowed to slowlyevaporate through pierced parafilm. Needle-like crystals were apparentafter ca. 2 weeks of evaporation.

Single Crystal X-Ray Diffraction: A lath-like needle of the sample wasselected for data collection. Diffraction data were collected with Mo-Kαradiation using a Bruker Smart Apex CCD diffractometer equipped with anOxford Cryosystems low-temperature device operating at 150 K.

On indexing the data set, the crystal structure was determined to bepseudosymmetric. Strong data could be indexed on a primitive, metricallymonoclinic, cell of dimensions a=3.76, b=30.23, c=12.12 Å, β=106.2°,V=1324 Å³. A complete indexing of all data could only be obtained with alarger triclinic cell of dimensions a=7.52, b=11.65, c=30.46 Å, α=89.8°,β=82.9°, γ=88.1°, V=2647 Å³. This cell is itself transformable to apseudo monoclinic C-centred cell of dimensions a=7.52, b=60.46, c=11.65Å, β=91.9°, V 5295 Å³.

The diffraction data were integrated and reduced (SAINT), and correctedfor systematic errors using the multiscan procedure SADABS. Thestructure was solved in P-1 by direct methods (SHELXS) using thedata-set integrated on the triclinic cell described above. The structurewas refined against |F|² using all data (SHELXL). Incorporation of atwin law was necessary for completion of the structure. The twin lawused was a two-fold rotation about the [−1 0 2] direction, whichcorresponds to the b-axis direction of the monoclinic cells describedabove.

In addition to being twinned, the structure is pseudosymmetric. Thismeans that the atomic coordinates within the organic fragments arerelated to each other, and it results in correlations and mathematicalinstabilities into the least squares refinement. In order to overcomethese similarities, restraints were applied to all chemically relatedbond distances and angles. Pairs of molecules (1 and 2, and 3 and 4) arerelated by a translation of a/2, and so equivalent anisotropicdisplacement parameters were constrained to be equal. Some damping wasneeded to achieve convergence. Correlation also causes equivalent bondlengths to become artificially different, and care should be taken notto ascribe any significance to apparent differences in chemicallyequivalent bond distances, for example. A more elaborate refinementmodel would be needed to resolve these effects.

Hydrogen atoms attached to carbon were placed in calculated positions.Some hydrogen atoms attached to oxygen could be located in differencemaps. In particular, the H-atoms were attached to the O-atoms ligatingthe sodium ions (O141 and O144). Positions for ligand-water H-atoms werelocated in a Fourier map calculated about the loci of possibleH-positions; those making geometrically sensible H-bonds and avoidingshort contacts were included in the model. H-atoms attached to O8 werelocated in a difference map, and the whole molecule then initiallyrefined as a rotating rigid group, thereafter the H-atoms were treatedwith a riding model. The remaining H-atoms (H7A and H142) were placedalong short O . . . O vectors. There was no evidence in Fourier maps forH-atoms on O42 and O43, and attempts to place them led to thedevelopment of unreasonably short H . . . H contacts with other H-atoms.

The final ‘conventional’ R-factor [based on F and 7355 data withF>4σ(F)] was 0.0616. Other crystal and refinement parameters are listedin Table 6.

TABLE 6 Single Crystal Data and Structure Refinement for the CrystallineGenistein Sodium Salt Dihydrate. A. CRYSTAL DATA Empirical formulaC₆₀H₅₄Na₂O₂₈, C₃₀H₃₂Na₂O₁₆, 2(C₁₅H₉O₅), 2(H₂O) Formula weight 1269.01Wavelength 0.71073 A Temperature 150(2) K Crystal system Triclinic Spacegroup P-1 Unit cell dimensions a = 7.524(2) A alpha = 89.762(7) deg. b =11.646(3) A beta = 82.902(10) deg. c = 30.464(6) A gamma = 88.073(10)deg. Volume 2647.4(12) A{circumflex over ( )}3 No. of reflections forcell 7324 (2.5 < theta < 25 deg.) Z 2 Density (calculated) 1.592Mg/m{circumflex over ( )}3 Absorption coefficient 0.141 mm{circumflexover ( )}−1 F(000) 1320 B. DATA COLLECTION Crystal description colorlessneedle-like lath Crystal size 0.70 × 0.16 × 0.10 mm Instrument BrukerSmart Apex CCD Theta range for data 0.67 to 24.55 deg. collection Indexranges −8 <= h <= 8, −13 <= k <= 13, −35 <= l <= 35 Reflectionscollected 33227 Independent reflections 8758 [R(int) = 0.0574] Scan typeomega Absorption correction Multiscan, (Tmin = 0.804, Tmax = 0.984) C.SOLUTION AND REFINEMENT Solution direct (SHELXS-97 (Sheldrick, 2008))Refinement type Full-matrix least-squares on F{circumflex over ( )}2Program used for SHELXL-97 refinement Hydrogen atom geom/difmapplacement Hydrogen atom treatment riding/rotating group Data/restraints/8758/1636/580 parameters Goodness-of-fit on F{circumflex over ( )}21.088 Conventional R R1 = 0.0616 [7355 data] [F > 4sigma(F)] Weighted RwR2 = 0.1489 (F{circumflex over ( )}2 and all data) Final maximum 0.073delta/sigma Weighting scheme calc w = 1/[\s{circumflex over( )}2{circumflex over ( )}(Fo{circumflex over ( )}2{circumflex over( )}) + (0.0598P){circumflex over ( )}2{circumflex over ( )}+ 2.2931P]where P = (Fo{circumflex over ( )}2{circumflex over ( )}+ 2Fc{circumflexover ( )}2{circumflex over ( )})/3 Largest diff. peak and 0.316 and−0.310 e · A{circumflex over ( )}−3 hole

Discussion: The single crystal structure of crystalline genistein sodiumsalt dihydrate shows that the compound has an overall formula of[Na₂(H₂O)₄(μ1-H₂O)₂(LH)₂]L₂.2H₂O where LH=the fully protonated genisteinligand C₁₅H₁₀O₅ and μ-H₂O are bridging water molecules between the Naions (i.e., the Na ions are each bonded to two terminal waters and twobridging waters (designated μ-H₂O), plus one LH ligand—see FIG. 9). Thisconclusion depends on the model of H-atom placement described above.Hydrogen atom placement using X-ray data is usually regarded astentative, the more so here because of the problems encountered duringstructure analysis. That being said, the H-atom positions proposed doform a plausible H-bonding set with all H-atoms involved ingeometrically normal hydrogen bonds.

As shown in FIG. 9, the cationic sodium complexes consist of dimericunits formed across inversion centres. The sodium ions arefive-coordinate, the coordination sphere consisting of two terminal andtwo bridging water ligands and one of the LH ligands. A hydrogen bond isformed between the ligating alcohol moiety and one of the terminal watermolecules (H141 . . . O1 and H141 . . . O4). The L⁻ anions aredeprotonated at the phenolic O42 and O43 sites. The C—O⁻ distances arequite short (average 1.34 Å). An internal hydrogen bond is formedbetween H6* and O8* the ligating LH and the L⁻ anions.

FIG. 9 illustrates the centrosymmetric disodium cation in the dimericstructure of crystalline genistein sodium salt dihydrate, wherein theintramolecular hydrogen bonds are shown as dashed lines.

The packing in the crystal is dominated by hydrogen bonding. The cationsare linked to the anions via water molecules to form layers which alsofeature stacking interactions between cations and anions. FIG. 10 showsone such layer involving cations based on O11 and anions based on O12.Water molecules are shown. The view is along [010].

Similar layers composed of molecules based on O13 and O14 are alsoformed, and the two types of layers alternate along the b-axis, beinglinked by H-bonds. FIG. 11 illustrates the overall picture as a threedimensional network. FIG. 11 illustrates the packing of crystallinegenistein sodium salt dihydrate viewed along the [100] direction.

Analysis using the PLATON/MISSYM procedure indicates that the organicfragments on their own can be described using the small (1324 Å³) celland space group P2₁/c, and it is only the sodium ions and watermolecules which break this symmetry, explaining the pattern of strongand weak data in the diffraction pattern, and the pseudosymmetryproblems experienced in refinement.

The calculated XRPD pattern based on the single crystal data andstructure for the crystalline genistein sodium salt dihydrate is shownin FIG. 12. Table 7 lists the peaks in the calculated XRPD pattern. Theentire list of peaks, or a subset thereof, may be sufficient tocharacterize crystalline genistein sodium salt dihydrate. One subset ofpeaks that, individually or in combination, may be used to characterizecrystalline genistein sodium salt dihydrate from FIG. 12 includes 5.8,11.6, 15.2, 17.6, 25.1, 28.4, 28.8, and 29.2 °2θ±0.2 °2θ.

TABLE 7 Pos. d-spacing Rel. Int. [°2θ] [Å] [%] 5.8 15.1 23.520 8.1 10.95.770 11.6 7.6 12.620 15.2 5.8 69.700 17.6 5.0 5.570 23.7 3.8 4.540 24.63.6 35.590 25.0 3.6 32.830 25.1 3.6 97.090 25.1 3.6 98.070 25.1 3.5100.000 25.2 3.5 73.730 25.3 3.5 14.020 25.3 3.5 9.220 25.7 3.5 7.90025.8 3.5 10.480 28.4 3.1 23.020 28.5 3.1 15.880 28.8 3.1 10.520 28.8 3.127.560 29.2 3.1 23.560 29.2 3.1 22.670 32.3 2.8 7.540

1.13 Bioavailability of Genistein Alone and From Crystalline GenisteinSodium Salt Dihydrate, Following Intraduodenal and IntravenousAdministration in Male Sprague-Dawley Rats.

Preparation of Dosing Solutions for In-Vivo Study: Genistein andCrystalline genistein sodium salt dihydrate were stored at roomtemperature under desiccant and protected from light. The dosingsolutions were prepared fresh from powders on the day of dosing. Thedosing solution for intravenous administration (IV) was prepared at 1mg/mL (free acid) in 50:50 DMSO:saline. The dosing solutions forintraduodenal administration (ID) were prepared at 2 mg/mL (genisteinfree acid) in a 0.2% sodium carboxymethyl cellulose (Na CMC) solution inwater.

Animal Dosing: The pharmacokinetics of genistein was evaluated in fastedmale Sprague-Dawley rats. Each animal was fitted with a jugular veincannula (JVC) for blood sampling. Animals intended for intravenousdosing were fitted with an additional JVC for dose administration.Animals intended for intraduodenal dosing were fitted with anintraduodenal cannula (IDC) for dose administration. Surgically modifiedanimals were housed one per cage. All animals were supplied with acommercial rodent diet (LabDiet, Certified Rodent Diet #5002) ad libitumprior to study initiation. Food was then withheld from the animals for aminimum of twelve hours before the study and during the study, untileight hours post dose when food was returned. Water was supplied adlibitum.

Intraduodenal dosing solutions were administered as a single bolus doseat time zero on the day of dosing. Intravenous doses were administeredas a slow IV injection over approximately 1 minute. Blood sampling timesbegan at the end of the infusion. Blood samples were collected. Thestudy design is shown in Table 8.

TABLE 8 Out-line of Comparative Pharmacokinetic Study of Genistein andCrystalline Genistein Sodium Salt Dihydrate, in Rats. Dosing SolutionDosing Blood Treatment Test Dosing Dose Conc Volume Sampling GroupCompound Route (mg/kg) (mg/ml) (ml/kg) Vehicle Time points 1 GenisteinID 20 10 2 0.2% Pre dose, NaCMC 15, 30 min, In water 1, 2, 3, 4, 6, 8and 24 h 2 Crystalline ID 20 10 2 0.2% Pre dose, Genistein NaCMC 15, 30min, Sodium In water 1, 2, 3, 4, 6, Salt 8 and 24 h Dihydrate 3Genistein IV 1 1 1 50% Pre dose, 2, DMSO 5, 15, 30 min, in saline 1, 2,3, 4, 8 and 24 h 4 Crystalline IV 1 1 1 50% Pre dose, 2, Genistein DMSO5, 15, 30 min, Sodium in saline 1, 2, 3, Salt 4, 8 and Dihydrate 24 h

Each blood sample was collected from the rats via a jugular vein cannulaand placed into chilled polypropylene tubes containing sodium heparin asan anticoagulant. Samples were centrifuged at a temperature of 4° C. andat a speed of 13,000 rpm for 5 minutes. Samples were maintained chilledthroughout processing. Each plasma sample was split into two aliquots.The first aliquot contained 50 μL of plasma. All remaining plasma volumewas used for the second aliquot. Samples were then placed on dry ice,and stored in a freezer set to maintain −60° C. to −80° C. The totalconcentration of genistein in plasma samples were analyzed by LC-MS/MSafter an overnight incubation with glucuronidase/arylsulfatase enzymemixture. Pharmacokinetic parameters were calculated using the WinNonlinsoftware.

Analysis of Plasma Samples: An LC-MS/MS analytical method for thedetermination of genistein in rat plasma was developed. Prior to sampleanalysis, a standard curve was analyzed to determine the specificity,range, and linearity of the method. Total genistein in plasma sampleswas determined by pre-treating all samples withβ-glucuronidase/arylsulfatase enzymes and incubating prior to analysis.Incubation with the enzyme mix deconjugated any glucuronide or sulfatemetabolites of genistein back to the parent form.

Acceptance Criteria for LC-MS/MS Analysis: One standard curve wasdispersed throughout each analytical run. At least ⅝ of the standardsmust be accurate to within ±20%, except at the LLOQ where ±25% isacceptable, in order for the run to pass.

Pharmacokinetic Analysis: Individual plasma concentrations versus timedata for genistein were subjected to non-compartmental analysis usingthe pharmacokinetic program WinNonlin v. 4.1. Plasma concentrationsbelow the limit of quantitation (10 ng/mL) were assigned a value of zerofor PK analysis only.

Results: As shown in FIG. 13, the mean plasma concentration and PKprofiles of genistein compared with crystalline genistein sodium saltdihydrate was markedly different, following ID dosing. The mean peakplasma concentration (C_(max)) of genistein from crystalline genisteinsodium dihydrate salt was 4.2 fold higher compared to the peak plasmaconcentration of genistein, 8330±2176 ng/mL and 1983±1130 ng/mL,respectively. Already within 15 minutes after ID dosing of crystallinegenistein sodium salt dihydrate maximum plasma concentration (C_(max))of genistein was observed, while the C_(max) of genistein was observedat 2 hours post dose (FIG. 13 and Table 10). The genisteinbioavailability from crystalline genistein sodium salt dihydrate was55±16% compared to 16±4.4% for genistein (Table 9).

TABLE 9 Pharmacokinetic Parameters after Intraduodenal Administration of20 mg/kg of Respective Form (mean ± SD, n = 3). Crystalline Genistein PKparameter Genistein Sodium Salt Dihydrate C_(max) (ng/ml) 1983 ± 11308330 ± 2176 t_(max) (h) 2.0 ± 0   0.83 ± 1.0  AUC_(last) (h · kg ·ng/ml/mg) 414 ± 111 1161 ± 358 Bioavailability (%)  16 ± 4.4 55 ± 16

As shown in Table 10, the pharmacokinetic profile of genistein andcrystalline genistein sodium salt dihydrate following IV dosing were notsignificantly different between the two forms.

TABLE 10 Pharmacokinetic Parameters after Intravenous Administration of1 mg/kg of Respective Form (mean ± SD, n = 3). Crystalline Genistein PKparameter Genistein Sodium Salt Dihydrate C₀ (ng/ml)¹ 6617 ± 1059 6640 ±1223 T_(1/2) (h) 1.4 ± 0.3 1.6 ± 0.9 CL (L/h/kg) 0.40 ± 0.09 0.47 ± 0.08Vss (L/kg) 0.40 ± 0.06 0.36 ± 0.09 AUC_(last) (h · kg · ng/ml/mg) 2533 ±638  2129 ± 331  AUC_(∞)(h · kg · ng/ml/mg) 2584 ± 639  2189 ± 356 ¹extrapolated to t = 0.

1.14 Physicochemical Characterization and the Kinetic and EquilibriumSolubility Comparison Between Genistein and Crystalline Genistein SodiumSalt Dihydrate.

Crystalline genistein sodium salt dihydrate shows superior early andlate intrinsic kinetic solubility profiles as compared to genistein inEtOH/dH₂0 solutions. The low late intrinsic kinetic solubility ofcrystalline genistein sodium salt dihydrate in 100% EtOH has lesspractical implications for the preclinical development given thenon-physiological nature of the solvent.

Experimental: Genistein and crystalline genistein sodium salt dihydratewere run in a SuperSol 1000 (PREVENTOR Gmbh) solubility assay and theconcentration of the compounds measured over time in a closed system bymeasuring the absorbance in a flow-through measuring chamber at awavelength of 250 nm. Since both compounds form suspensions in puredeionized H₂O, physicochemical properties were assessed from solutionsof 100% EtOH as well as mixtures of dH₂O and EtOH, specifically, EtOH50/50 (vol/vol) and EtOH/dH₂O 75/25 (vol/vol) according to EuropeanPharmacopeia guidelines 01/2008, Section 2.9.3., Table 2.9.3.5.

The following parameters were measured:t_(|MSS|) defined as: Time from start of analysis to MaximumSolubilization Speed (min).C_(|MSS|) defined as: Early kinetic solubility as expressed asconcentration at Maximum Solubilization Speed (mg×1⁻¹).C_(|Eq|) defined as: Late kinetic solubility as expressed asconcentration at Equilibrium Kinetic Solubility (mg×1⁻¹).t_(|Eq|) defined as: Time from start of analysis to Equilibrium Kineticsolubility (min).ΔC[C_(Eq)−C_(MSS)] defined as: Difference in concentration between Earlyand Late Kinetic Solubility as defined above (mg×1⁻¹).Δt[C_(Eq)−C_(MSS)] defined as: Difference in time between Early and LateKinetic Solubility endpoints (min).MSS defined as: Maximum Solubility Speed defined by C_([MSS])/t_([MSS])(mg×1⁻¹×min⁻¹).ISI defined as: Intrinsic Solubility Index defined byΔC[C_(Eq)−C_(MSS)]/Δt[C_(Eq)−C_(MSS)].The higher the ISI value, the faster the solubilization and the strongerthe relative contribution of the late, intrinsic kinetic equilibriumsolubility C_([eq]).KSR defined as: Kinetic Solubility ratio given by C_([MSS])/C_([Eq]).The KSR is the numerical ratio indicator of the relative contribution ofthe early kinetic Solubility to the Overall Late Kinetic EquilibriumSolubility. The higher the KSR Value, the stronger the relativecontribution of the early kinetic solubility C_([MSS]).

Results: The thermodynamic kinetic and equilibrium solubility data ofgenistein and crystalline genistein sodium salt dihydrate were assessedunder the conditions reported in Tables 11, 12, and 13.

As shown in Table 11, genistein showed (a) good MSS, (b) acceptable KSRand (c) good late solubility profiles, while crystalline genisteinsodium salt dihydrate showed (a) excellent MSS (b) excellent KSR and (c)good-to-acceptable ISI. For EtOH/dH₂O 50/50 (vol/vol) crystallinegenistein sodium salt dihydrate displayed the best early intrinsickinetic solubility profile.

TABLE 11 EtOH/dH₂O 50/50 (vol/vol) ΔC Δt [C_(Eq) − [C_(Eq) − MSSt_([MSS]) C_([MSS]) C_([Eq]) t_([Eq]) C_(MSS)] C_(MSS)] ISI KSRGenistein 12.18 0:61 7.43 12.87 4:12 5.44 3:51 1.55 0.58 Crystalline20.66 0:71 14.67 17.40 5:28 2.73 4:57 0.60 0.84 Genistein Sodium SaltDihydrate

For EtOH/dH₂O 75/25 (vol/vol), as shown in Table 13, genistein showed(a) good MSS, (b) good KSR and (c) good late solubility profiles.Crystalline genistein sodium salt dihydrate showed (a) excellent MSS (b)excellent KSR and (c) excellent ISI, which is the best early and lateintrinsic kinetic solubility profile.

TABLE 12 EtOH/dH₂O 75/25 (vol/vol) ΔC Δt [C_(Eq) − [C_(Eq) − MSSt_([MSS]) C_([MSS]) C_([Eq]) t_([Eq]) C_(MSS)] C_(MS)] ISI KSR Genistein19.67 0:44 8.52 14.03 4:15 5.51 3:71 1.49 0.61 Crystalline 37.33 0:3011.20 15.86 4:01 4.66 3:71 1.26 0.71 Genistein Sodium Salt Dihydrate

As reported in Table 13, at EtOH 100%, genistein showed (a) good MSS,(b) acceptable KSR and (c) good late solubility profiles, whilecrystalline genistein sodium salt dihydrate, in comparison, showed (a)excellent MSS (b) excellent KSR and (c) poor ISI. Crystalline genisteinsodium salt dihydrate showed the best early intrinsic kinetic solubilityprofile, but small contribution to the overall profile.

TABLE 13 EtOH 100% ΔC Δt [C, − [C_(Eq) − MSS t_([MSS]) C_([MSS])C_([Eq]) t_([Eq]) C_(MSS)] C_(MS)] ISI KSR Genistein 23.81 0:24 5.8113.79 4:03 7.98 3:79 2.11 0.42 Crystalline 36.90 0:29 10.85 11.20 5:140.35 4:85 0.07 0.97 Genistein Sodium Salt Dihydrate

1.15 Large Scale Synthesis of Crystalline Genistein Sodium SaltDihydrate

Synthesis: Crystalline genistein sodium salt dihydrate was prepared on akilogram scale using the following procedure:

-   -   1. 5.2 kg of 2-propanol (IPA) and 320 g of neutral genistein        were charged into a 15 L glass reactor.    -   2. The temperature of the mixture was adjusted to 22±3° C. and        632 g of 2M aq. NaOH was added dropwise during about 40 minutes        at 22±4° C.    -   3. The mixture was agitated at 22±4° C. for about 19 hours and        cooled to about 15° C. and agitated for 4 hours.    -   4. The mixture was agitated at temperature cycles (15±3°        C.→35±3° C. during 1 h→35±3° C. for 4 h→15±3° C. during 1 h        →15±3° C. for 4 h) for about 90 hours and finally at 15±3° C.        for about 4.5 h.    -   5. The precipitated product was filtered and washed with 1.2 kg        of pre-cooled 2-propanol.    -   6. The filtered product was dried in vacuum tray dryer without        vacuum at first at the set temperature of 30° C. for about 19 h,        then at the set temperature of 40° C. for about 20 h, then at        the set temperature of 50° C. for about 24 h, then at the set        temperature of 60° C. for about 16 h and finally at set        temperature of 70° C. for about 10 h until the water content        measured by KF-titration met set specification.    -   7. Finally, the product (0.24 kg) was ground and packed into        PE-bags.

Optional Recrystallization Procedure Crystalline genistein sodium saltdihydrate was recrystallized using the following procedure:

-   -   1.24 g of the crystalline genistein sodium salt dihydrate        prepared as above was added to 240 ml of ethanol.    -   2. This mixture was stirred at 250 rpm and heat at 45° C., for        ca. 30 min.    -   3. The resulting solution was allowed to cool to room        temperature.    -   4. Heptane was then added in aliquots (detailed as follows)        adding 1 aliquot per 1 min. Intermittent stirring at 40 rpm was        used between each addition.        -   Added 4.151 ml of heptane and stirred intermittently at 40            rpm.        -   Added 3.272 ml of heptane and stirred intermittently at 40            rpm.        -   Added 5.209 ml of heptane and stirred intermittently at 40            rpm.        -   Added 3.505 ml of heptane and stirred intermittently at 40            rpm.        -   Added 3.885 ml of heptane and stirred intermittently at 40            rpm.        -   Added 5.465 ml of heptane and stirred intermittently at 40            rpm.        -   Added 6.314 ml of heptane and stirred intermittently at 40            rpm.        -   Added 6.656 ml of heptane and stirred intermittently at 40            rpm.        -   Added 8.258 ml of heptane and stirred intermittently at 40            rpm.        -   Added 6.969 ml of heptane and stirred intermittently at 40            rpm.        -   Added 11.115 ml of heptane and stirred intermittently at 40            rpm.        -   Added 10.750 ml of heptane and stirred intermittently at 40            rpm.        -   Added 14.219 ml of heptane and stirred intermittently at 40            rpm.        -   Added 9.261 ml of heptane and stirred intermittently at 40            rpm.        -   Added 14.913 ml of heptane and stirred intermittently at 40            rpm.        -   Added 13.471 ml of heptane and stirred intermittently at 40            rpm.        -   Added 15.753 ml of heptane and stirred intermittently at 40            rpm.        -   Added 19.172 ml of heptane and stirred intermittently at 40            rpm.        -   Added 23.441 ml of heptane and stirred intermittently at 40            rpm.        -   Added 25.503 ml of heptane and stirred intermittently at 40            rpm.        -   Added 26.856 ml of heptane and stirred intermittently at 40            rpm.        -   Added 28.126 ml of heptane and stirred intermittently at 40            rpm.        -   Added 28.070 ml of heptane and stirred intermittently at 40            rpm.        -   Added 36.738 ml of heptane and stirred intermittently at 40            rpm.        -   Added 35.989 ml of heptane and stirred intermittently at 40            rpm.        -   Added 49.677 ml of heptane and stirred intermittently at 40            rpm.        -   Added 50.145 ml of heptane and stirred intermittently at 40            rpm.        -   Added 32.579 ml of heptane and stirred intermittently at 40            rpm.        -   Added 61.538 ml of heptane and stirred intermittently at 40            rpm.        -   Added 57.143 ml of heptane and stirred intermittently at 40            rpm.        -   Added 51.948 ml of heptane and stirred intermittently at 40            rpm.        -   Added 90.909 ml of heptane and stirred intermittently at 40            rpm.    -   5. The sample was then left to crystallize overnight at room        temperature (ca. 18 hrs).    -   6. The crystalline product was collected by vacuum filtration.    -   7. The crystalline product was then dried for ca. 21 hours while        monitoring the water content by Karl Fischer titration to avoid        the risk of dehydration.

FIG. 14 shows the XRPD pattern of the recrystallized crystallinegenistein sodium salt dihydrate. The peaks in the XRPD pattern at anexperimental °2θ±0.2 °2θ are listed in Table 14. The entire list ofpeaks, or a subset thereof, may be sufficient to characterizecrystalline genistein sodium salt dihydrate. One subset of peaks that,individually or in combination, may be used to characterize crystallinegenistein sodium salt dihydrate from FIG. 14 includes 6.0, 7.1, 11.8,11.9, 15.3, 17.8, 21.3, 25.0, 28.3, 28.6, and 29.1 °2θ±0.2 °2θ.Preferred subset of peaks includes 6.0, 7.1, 15.3, and 25.0, and atleast two of the three peaks 28.3, 28.6, and 29.1 °2θ±0.2 °2θ, andanother preferred subset of peaks includes 6.0, 7.1, 15.3, 25.0, and28.3 °2θ±0.2 °2θ.

TABLE 14 Pos. [°2θ ± 0.2 °2θ] d-spacing [Å] Rel. Int. [%] 6.0 14.6 33.537.1 12.4 21.77 8.3 10.7 5.53 11.8 7.5 15.05 11.9 7.4 12.04 15.3 5.890.11 15.6 5.7 5.91 17.8 5.0 17.38 19.3 4.6 7.99 21.3 4.2 10.78 22.1 4.04.67 23.4 3.8 5.61 23.7 3.8 9.49 24.5 3.6 35.37 25.0 3.6 100 25.4 3.59.09 25.6 3.5 16.02 25.8 3.5 18.24 27.6 3.2 6.76 28.3 3.2 21.60 28.6 3.122.15 29.1 3.1 20.45 29.9 3.0 5.12 30.7 2.9 12.80 30.8 2.9 9.80 32.1 2.85.32 35.4 2.5 8.63 35.7 2.5 8.78 36.6 2.5 5.38 39.1 2.3 5.66 40.5 2.26.66 41.0 2.2 8.72 41.1 2.2 7.29 41.9 2.2 8.62 42.6 2.1 8.97

1. Genistein sodium salt dihydrate.
 2. Crystalline genistein sodium salt dihydrate characterized by an XRPD pattern having peaks at 6.0, 7.1, 15.3, and 25.0 °2θ±0.2 °2θ, and at least two of the three peaks 28.3, 28.6, and 29.1 °2θ±0.2 °2θ.
 3. Crystalline genistein sodium salt dihydrate characterized by an XRPD pattern having peaks at 6.0, 7.1, 15.3, 25.0, and 28.3 °2θ±0.2 °2θ.
 4. A therapeutic composition comprising a therapeutically effective amount of genistein sodium salt dihydrate according to claim 1 and at least one pharmaceutically acceptable carrier.
 5. A method of treating cancer comprising the step of administering to a patient in need thereof a therapeutically effective amount of a therapeutic composition of claim
 4. 6. A method of treating cancer comprising the step of administering to a patient in need thereof a therapeutically effective amount of genistein sodium salt dihydrate according to claim
 1. 7. A method of treating chronic inflammation (inflammatory diseases) comprising the step of administering to a patient in need thereof a therapeutically effective amount of a therapeutic composition of claim
 4. 8. A method of treating chronic inflammation (inflammatory diseases) comprising the step of administering to a patient in need thereof a therapeutically effective amount of genistein sodium salt dihydrate according to claim
 1. 9. A method of treating transthyretin amyloidoses comprising the step of administering to a patient in need thereof a therapeutically effective amount of a therapeutic composition of claim
 4. 10. A method of treating transthyretin amyloidoses comprising the step of administering to a patient in need thereof a therapeutically effective amount of genistein sodium salt dihydrate according to claim
 1. 11. A method of treating cystic fibroses comprising the step of administering to a patient in need thereof a therapeutically effective amount of a therapeutic composition of claim
 4. 12. A method of treating cystic fibroses comprising the step of administering to a patient in need thereof a therapeutically effective amount of genistein sodium salt dihydrate according to claim
 1. 13. A method of treating infection comprising the step of administering to a patient in need thereof a therapeutically effective amount of a therapeutic composition of claim
 4. 14. A method of treating infection comprising the step of administering to a patient in need thereof a therapeutically effective amount of genistein sodium salt dihydrate according to claim
 1. 15. A therapeutic composition comprising a therapeutically effective amount of crystalline genistein sodium salt dihydrate according to claim 2 and at least one pharmaceutically acceptable carrier.
 16. A method of treating cancer comprising the step of administering to a patient in need thereof a therapeutically effective amount of a therapeutic composition of claim
 15. 17. A method of treating cancer comprising the step of administering to a patient in need thereof a therapeutically effective amount of crystalline genistein sodium salt dihydrate according to claim
 2. 18. A method of treating chronic inflammation (inflammatory diseases) comprising the step of administering to a patient in need thereof a therapeutically effective amount of a therapeutic composition of claim
 15. 19. A method of treating chronic inflammation (inflammatory diseases comprising the step of administering to a patient in need thereof a therapeutically effective amount of crystalline genistein sodium salt dihydrate according to claim
 2. 20. A method of treating transthyretin amyloidoses comprising the step of administering to a patient in need thereof a therapeutically effective amount of a therapeutic composition of claim
 15. 21. A method of treating transthyretin amyloidoses comprising the step of administering to a patient in need thereof a therapeutically effective amount of crystalline genistein sodium salt dihydrate according to claim
 2. 22. A method of treating cystic fibroses comprising the step of administering to a patient in need thereof a therapeutically effective amount of a therapeutic composition of claim
 15. 23. A method of treating cystic fibroses comprising the step of administering to a patient in need thereof a therapeutically effective amount of crystalline genistein sodium salt dihydrate according to claim
 2. 24. A method of treating infection comprising the step of administering to a patient in need thereof a therapeutically effective amount of a therapeutic composition of claim
 15. 25. A method of treating infection comprising the step of administering to a patient in need thereof a therapeutically effective amount of crystalline genistein sodium salt dihydrate according to claim
 2. 